CN1860228A - Staple type oligonucleotide and drug comprising the same - Google Patents

Staple type oligonucleotide and drug comprising the same Download PDF

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CN1860228A
CN1860228A CNA2004800285477A CN200480028547A CN1860228A CN 1860228 A CN1860228 A CN 1860228A CN A2004800285477 A CNA2004800285477 A CN A2004800285477A CN 200480028547 A CN200480028547 A CN 200480028547A CN 1860228 A CN1860228 A CN 1860228A
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oligonucleotide
staple type
medicine
bases
type oligonucleotide
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CN1860228B (en
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椚座康夫
富田奈留也
桥本英雄
吉川秀树
森下篭一
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Anges Inc
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Anges MG Inc
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Abstract

Conventional oligonucleotides are opened at both ends and thereby unstable. The stability of them against catabolic enzymes is increased by phosphorothioate modification, but such phosphorothioate causes toxicity. The present invention provides oligonucleotides and medicaments in which these problems are improved. That is, it provides a staple oligonucleotides and medicaments containing the same as the active ingredient. Specifically, it provides transcription factor inhibitors, antisense oligonucleotides and siRNAs. More specifically, it provides agents for preventing, treating or improving inflammation, autoimmune diseases, central diseases, reperfusion injury in ischaemic diseases, worsened prognosis after organ transplantation or organ surgery, or restenosis after PTCA. Further specifically, it provides agents for preventing, treating or improving arthritis, dermatitis, nephritis, hepatitis, renal failure, cystitis, prostatitis, urethritis, ulcerative colitis, Crohn disease, chronic rheumatoid arthritis, osteoarthritis, atopic dermatitis, contact dermatitis, psoriasis, cutaneous ulcer or decubitus.

Description

Staple type oligonucleotide and comprise the medicine of this staple type oligonucleotide
Technical field
The present invention relates to novel staple (staple) type oligonucleotide and with its medicine as effective constituent.
Background technology
Oligonucleotide in the past is widely used as transcription factor inhibitor, antisense oligonucleotide, siRNA etc.
Wherein, for example as the transcription factor inhibitor, can list molecule bait (the bait oligonucleotide is hereinafter referred to as bait) type nucleic acid particularly, it hinders the activity of the transcription factor of controlling gene expression specifically.
Transcribing here is meant when biological intravital genetic information is expressed, and is the process of the synthetic messenger RNA(mRNA) of template with DNA, being basic synthetic protein by the information of transcribing the messenger RNA(mRNA) of making.This factor of transcribing of control is called transcription regulaton factor.
Specifically, known have 54 kinds of NF-κ B3, STAT-1, STAT-2, STAT-3, STAT-4, STAT-5, STAT-6, GATA-3, AP-1, E2F, Ets, CRE etc.
As antisense oligonucleotide, can list the medicine that has the sequence that is involutory with target gene and suppress this genetic expression particularly.
As siRNA, can list particularly by RNA and interfere (RNA interferance; RNAi) hinder the medicine that target gene is expressed.
In addition, being characterized as of these oligonucleotide structurally constitutes two strands.
The existing document that becomes background of the present invention is: Biochem Biophys Res Commun.2003 Sep5; 308 (4): 689-97, Gene Ther.2002 Dec; 9 (24): 1682-92 and CircRes.2002 Jun 28; 90 (12): 1325-32.
Summary of the invention
Problem points to be solved by this invention is: the oligonucleotide of type was because two ends were to open (open), therefore instability in the past; In addition, though modify the stability that improves exonuclease lytic enzymes such as (exonuclease), cause toxic generation by thiophosphatephosphorothioate by phosphorothioate (Sization).
The present invention is specially following material and medicine.
(1) staple type oligonucleotide, it is a single stranded oligonucleotide, wherein, 5 ' terminal sequence has and the reciprocal complementarity of pars intermedia sequence, 3 ' terminal sequence also has and the reciprocal complementarity of pars intermedia sequence, the two ends of pars intermedia have intramolecularly do not form complementary in conjunction with and comprise the ring portion of 3～10 base sequences.
(2) as (1) described staple type oligonucleotide, wherein, single stranded oligonucleotide is that 30～70 bases are long.
(3) as (1) or (2) described staple type oligonucleotide, wherein, single stranded oligonucleotide is that 34～64 bases are long.
(4) as each described staple type oligonucleotide of (1)～(3), wherein, single stranded oligonucleotide is that 38～58 bases are long.
(5) as each described staple type oligonucleotide of (1)～(4), wherein single stranded oligonucleotide is that 42～54 bases are long.
(6) as each described staple type oligonucleotide of (1)～(5), wherein ring portion is that 4～6 bases are long.
(7) as each described staple type oligonucleotide of (1)～(6), wherein single stranded oligonucleotide is that 42～54 bases are long, ring portion is that 4～6 bases are long.
(8) as each described staple type oligonucleotide of (1)～(7), wherein oligonucleotide is DNA or DNA derivative.
(9) as each described staple type oligonucleotide of (1)～(8), it is characterized in that phosphate is not by phosphorothioate.
(10) as each described staple type oligonucleotide of (1)～(9), it is a kind that is selected from in the oligodeoxynucleotide shown in the sequence number 1～3 of sequence table.
(11) comprise (1)～medicine of (10) each described staple type oligonucleotide.
(12) as (11) described medicine, this medicine is transcription factor inhibitor, antisense oligonucleotide or siRNA.
(13) as (12) described medicine, wherein the transcription factor inhibitor is an antagonism type inhibitor.
(14) as (12) or (13) described medicine, wherein transcription factor is a kind that is selected among NF-κ B, STAT-1, STAT-2, STAT-3, STAT-4, STAT-5, STAT-6, GATA-3, AP-1, E2F, Ets and the CRE.
(15) as each described medicine of (12)～(14), this medicine be inflammation, anaphylactic disease, autoimmune disorder, central disease, ischemic disease pour into prognosis deterioration, percutaneous transluminal coronary angioplasty (percutaneoustransluminal coronary angioplasty behind obstacle, organ transplantation or the organ surgery again; The prevention of the restenosis PTCA), treatment or activator.
(16) as each described medicine of (12)～(15), wherein inflammation is sacroiliitis, dermatitis, ephritis, hepatitis, renal failure, urocystitis, prostatitis, urethritis, ulcerative colitis or Crohn disease (Crohn disease).
(17) as (16) described medicine, wherein sacroiliitis is chronic rheumatoid arthritis or arthritis deformans.
(18) as (16) described medicine, wherein dermatitis is atopic dermatitis, contact dermatitis, chronic eczema, skin ulcer or bedsore.
(19) each described staple type oligonucleotide of (1)～(10) is used to make the purposes of transcription factor inhibitor, antisense oligonucleotide or siRNA.
(20) each described staple type oligonucleotide of (1)～(10) by take significant quantity on the pharmacology to the patient, thus prevent, treat or improve transcription factor inhibitor, antisense oligonucleotide or siRNA method to its effective disease.
Staple type oligonucleotide among the present invention is a strand, it has following staple type structure (shape of the staple type after the extruding), promptly 5 ' terminal sequence has and the reciprocal complementarity of pars intermedia sequence, 3 ' terminal sequence also has and the reciprocal complementarity of pars intermedia sequence, the two ends of pars intermedia have intramolecularly do not form complementary in conjunction with and comprise the ring portion of 3～10 base sequences, particularly, the structure that for example has following chemical formula.
Figure A20048002854700081
In the formula, vertical line is represented non-binding [5 ' end and 3 ' end].
In addition, its chain length is unqualified, and it is long to be generally 30～70 bases, and it is long to be preferably 34～64 bases, and more preferably 38～58 bases are long, and more preferably 42～54 bases are long.
Ring portion is that 3～10 bases are long, and it is long to be preferably 4～6 bases.
The chain length of the reflex part sequence of 5 ' end and 3 ' end (since 5 ' end or 3 ' end sequence till the ring portion, that have complementarity) is also unqualified, and it is long to be generally 4～20 bases, and it is long to be preferably 6～18 bases, and more preferably 8～16 bases are long.
The chain length of the reflex part sequence of 5 ' end and 3 ' end can identical (symmetric figure), also can difference (asymmetric shape).
Oligonucleotide among the present invention is unqualified, can be DNA, DNA derivative, RNA or RNA derivative, but more preferably DNA or DNA derivative.
As the concrete example of staple type oligonucleotide of the present invention, for example can list with the oligodeoxynucleotide shown in the sequence number 1～3 of sequence table.
Staple type oligonucleotide of the present invention can be according to well-established law, after utilizing synthetic target single stranded sequence such as DNA synthesizer, and heating in solvent and obtaining.
Thiophosphatephosphorothioate among the present invention is meant part or all structure that is replaced by sulphur atom of the Sauerstoffatom in the phosphate.
The pharmaceutical use of the staple type oligonucleotide among the present invention is unqualified, specifically, for example be transcription factor inhibitor, antisense oligonucleotide, siRNA etc., more particularly, for example for inflammation, autoimmune disorder, central disease, ischemic disease pour into again that prognosis behind obstacle, organ transplantation or the organ surgery worsens, prevention, treatment or the activator of restenosis behind the PTCA etc.
The inflammation here more specifically can list for example sacroiliitis, dermatitis, ephritis, hepatitis, renal failure, urocystitis, prostatitis, urethritis, ulcerative colitis, Crohn disease etc.
Secondly, the sacroiliitis here more specifically can list for example chronic rheumatoid arthritis (RA) or arthritis deformans (OA) etc.
Moreover, as dermatitis, more specifically can list for example atopic dermatitis, contact dermatitis, chronic eczema, skin ulcer, bedsore etc.
The dosage of the staple type oligonucleotide among the present invention or route of administration are also according to the difference of the kind of disease or degree, symptom, patient age, sex, complication, coupling medicine etc. and difference, do not limit, but be generally each 10 μ g～10g, be preferably 100 μ g～5g, 1mg～1g more preferably, by through skin, subcutaneous, intraarticular, intramuscular, intravenously or oral administration.
Except that the present invention, for example also have disclosed ring-type bait (dumbbell shape bait) in the WO03/091432 communique etc., but staple type of the present invention has open loop portion, different fully on the structure.
By enforcement of the present invention, the unstable that had of type oligonucleotide was enhanced in the past, can reduce dosage, and can also improve security.
Description of drawings
Fig. 1 is for representing the LPS stimulation after 24 hours, the figure of the IL-1 β amount in the culture supernatant.
Fig. 2 is for representing the LPS stimulation after 24 hours, the figure of the IL-1 β amount in the synovial membrane supernatant.
Fig. 3 is the electrophorogram of the stability of expression staple type bait.
Embodiment
Below, enumerating embodiment and further describe the present invention, the present invention is not subjected to the qualification of these embodiment certainly.
Embodiment 1
The antiphlogistic effects research of staple type oligonucleotide
A. cytokine is quantitative
1. the processing of synovial tissue
Behind synovial tissue's homogeneous of the patient with rheumatoid arthritis of gathering in the time of (1) will performing the operation, every 100mg is inoculated in (serum free medium 500 μ l/ holes) in 24 orifice plates.
The transfection of NF-kappa B decoy, Scramble bait (HVJ envelope method)
(2) in HVJ1.1 * 10 4HAU/1.1ml BSS[balanced salt solution (10mM Tris-HCl, pH are 7.6 for 137mM NaCl, 5.4mM KCl)] state under with 99mJ/cm 2Carry out UV treatment.
(3) every 1ml is sub-packed in the 1.5ml pipe, at 4 ℃, and centrifugal treating 15min under the condition of 15000rpm.
(4) in the bait of 200 μ g, add BSS, be 92 μ l.
(5) add 8 μ l 3%TritonX-100/TE buffered soln.
(6), behind the centrifugal 15min, remove supernatant under the condition of 15000rpm at 4 ℃.
(7) after interpolation 1ml BSS mixes, centrifugation 15min under the condition of 15000rpm.
(8) remove supernatant after, be suspended among the PBS of 200 μ l.
(9) in synovial tissue, add bait-HVJ envelope mixture, be 15 μ M, and in 37 ℃ CO2gas incubator, cultivated 30 minutes.
Add the sequence of bait
Double-stranded NF-kappa B decoy
5 '-CCTTGAAGGGATTTCCCTCC-3 '/5 '-GGAGGGAAATCCCTTCAAGG-3 ' (two strands)
The Scramble bait
5 '-CATGTCGTCACTGCGCTCAT-3 '/5 '-ATGAGCGCAGTGACGACATG-3 ' (two strands)
Staple type oligonucleotide (i)
5 '-ATTTCCCTCCAAAAGGAGGGAAATCCCTTCAAGGAAAACCTTGAAGGG-3 ' (connecting) at 1 place
The dumbbell shape oligonucleotide (ii)
5 '-ATTTCCCTCCAAAAGGAGGGAAATCCCTTCAAGGAAAACCTTGAAGGG-3 ' (connecting) at 2 places
2.LPS stimulate
(10) remove bait-HVJ envelope mixture, add the nutrient solution (substratum) that 500 μ l contain 10%FBS, add LPS, be 0.01 μ g/ml.
3. the mensuration of the recovery of nutrient solution, synovial tissue, IL-1 β
Reclaim nutrient solution and synovial tissue after (11) 24 hours.In synovial tissue, add 500 μ lPBS, use clarifixator to homogenize.
Behind the centrifugal 10min, collect supernatant under the condition of 5000rpm.It is preceding-20 ℃ of preservations to measure IL-1 β.
(12) use IL-1 β ELISA test kit (ENDOGEN company, catalog number (Cat.No.): EH21L1B) measure culture supernatant, synovial membrane supernatant.
4. result
IL-1 β amount (pg/ml) (with reference to Fig. 1) in the culture supernatant
NT(LPS0) SC(LPS0) NF(LPS0) R1(LPS0) R2(LPS0) 90.8 49.6 102.1 14.6 22.9 NT(LPS0.01) SC(LPS0.01) NF(LPS0.01) R1(LPS0.01) R2(LPS0.01) 303.9 370.7 312.6 25.1 74.3
IL-1 β amount (pg/ml) (with reference to Fig. 2) in the synovial membrane supernatant
NT(LPS0) SC(LPS0) NF(LPS0) R1(LPS0) R2(LPS0) 17.5 7.2 10.5 13.5 15.6 NT(LPS0.01) SC(LPS0.01) NF(LPS0.01) R1(LPS0.01) R2(LPS0.01) 170.9 145.7 484.8 38.9 111.2
NT: untreated fish group
SC:scramble bait administration group
NF:NF kappa B decoy administration group
R1: staple type oligonucleotide (connection of 1 place)
R2: dumbbell shape oligonucleotide (connection of 2 places)
In staple type oligonucleotide effect group, suppressed the generation of the IL-1 β in culture supernatant, the synovial membrane supernatant.The inhibition effect of the staple type oligonucleotide that 1 place connects is stronger.(a little less than the inhibition effect of double-stranded NF κ B effect group in this experiment.)
The quantitative whole flow process of above-mentioned cytokine is as follows.
Figure A20048002854700121
Embodiment 2
B. the stability test of dumbbell shape bait (with reference to Fig. 3)
Purpose: the patience of the bait in synovial fluid (stoste) relatively
Sequence, experiment condition:
1) the double-stranded bait of Sization
2) Sization staple type bait
3) non-Sization staple type bait (no Sization)
4) strand bait (the connection last stage of staple type oligonucleotide)
5) terminal Sization strand bait (the connection last stage of staple type oligonucleotide, only the bait of two terminal Sization)
To the synovial fluid (stoste) that wherein adds 0%, 50% or 100%, utilize the more stable property of electrophoresis respectively.
Result: in synovial fluid, 1) Sization staple type bait and the 5 double-stranded bait of Sization, 2)) terminal Sization strand bait is stable, 3) non-Sization staple type bait is basicly stable, 4) the strand bait is decomposed.
In detail, 1) the double-stranded bait of Sization and 2) Sization staple type bait, even also all similarly stable in 100% synovial fluid with 0% (do not have and add).
In addition, 3) stability of non-Sization staple type bait and the minimizing of the concentration dependent ground of synovial fluid, even but in 100% synovial fluid, also have the stable bait that can detect completely.
On the other hand, 4) strand bait terminal Sization strand bait and 5) is with 1)～3) compare stable low in synovial fluid.
But, if relatively 4) and terminal Sization strand bait and 5) the strand bait, as can be known 4) even terminal Sization strand bait also has micro-stable bait in 100% synovial fluid, and 5) even the strand bait does not have stable bait yet in 50% synovial fluid.
Sequence table
＜110〉Anges Mg Inc
＜120〉staple type oligonucleotide and comprise the medicine of this staple type oligonucleotide
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<150>JP 2003-341419
<151>2003-09-30
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Claims (20)

1. staple type oligonucleotide, it is a single stranded oligonucleotide, wherein 5 ' terminal sequence has and the reciprocal complementarity of pars intermedia sequence, 3 ' terminal sequence also has and the reciprocal complementarity of pars intermedia sequence, the two ends of pars intermedia have intramolecularly do not form complementary in conjunction with and comprise the ring portion of 3～10 base sequences.
2. staple type oligonucleotide as claimed in claim 1, wherein, single stranded oligonucleotide is that 30～70 bases are long.
3. staple type oligonucleotide as claimed in claim 1 or 2, wherein, single stranded oligonucleotide is that 34～64 bases are long.
4. as each described staple type oligonucleotide of claim 1～3, wherein, single stranded oligonucleotide is that 38～58 bases are long.
5. as each described staple type oligonucleotide of claim 1～4, wherein, single stranded oligonucleotide is that 42～54 bases are long.
6. as each described staple type oligonucleotide of claim 1～5, wherein, ring portion is that 4～6 bases are long.
7. as each described staple type oligonucleotide of claim 1～6, wherein, single stranded oligonucleotide is that 42～54 bases are long, ring portion is that 4～6 bases are long.
8. as each described staple type oligonucleotide of claim 1～7, wherein, oligonucleotide is DNA or DNA derivative.
9. as each described staple type oligonucleotide of claim 1～8, it is characterized in that phosphate is not by phosphorothioate.
10. as each described staple type oligonucleotide of claim 1～9, it is a kind that is selected from in the sequence number 1～3 of sequence table or the oligodeoxynucleotide shown in the following structural formula,
In the formula, vertical line is represented non-binding [5 ' end and 3 ' end].
11. comprise the medicine of each described staple type oligonucleotide of claim 1～10.
12. medicine as claimed in claim 11, this medicine are transcription factor inhibitor, antisense oligonucleotide or siRNA.
13. medicine as claimed in claim 12, wherein, the transcription factor inhibitor is an antagonism type inhibitor.
14. as claim 12 or 13 described medicines, wherein transcription factor is a kind that is selected among NF-κ B, STAT-1, STAT-2, STAT-3, STAT-4, STAT-5, STAT-6, GATA-3, AP-1, E2F, Ets and the CRE.
15. as each described medicine of claim 12～14, this medicine be inflammation, anaphylactic disease, autoimmune disorder, central disease, ischemic disease pour into the prognosis deterioration behind obstacle, organ transplantation or the organ surgery, prevention, treatment or the activator of the restenosis after the percutaneous transluminal coronary angioplasty again.
16. as each described medicine of claim 12～15, wherein, inflammation is sacroiliitis, dermatitis, ephritis, hepatitis, renal failure, urocystitis, prostatitis, urethritis, ulcerative colitis or Crohn disease.
17. medicine as claimed in claim 16, wherein, sacroiliitis is chronic rheumatoid arthritis or arthritis deformans.
18. medicine as claimed in claim 16, wherein, dermatitis is atopic dermatitis, contact dermatitis, chronic eczema, skin ulcer or bedsore.
19. each described staple type oligonucleotide of claim 1～10 is used to make the purposes of transcription factor inhibitor, antisense oligonucleotide or siRNA.
20., thereby prevent, treat or improve transcription factor inhibitor, antisense oligonucleotide or siRNA method to its effective disease by each described staple type oligonucleotide of claim 1～10 of taking significant quantity on the pharmacology to the patient.
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Publication number Priority date Publication date Assignee Title
GB0404209D0 (en) * 2004-02-25 2004-03-31 Uws Ventures Ltd Materials and methods for treatment of allergic disease
US20080311552A1 (en) * 2005-09-20 2008-12-18 London Health Sciences Centre Research, Inc. Use of Sirnas in Organ Storage/Reperfusion Solutions
JP5601777B2 (en) * 2008-02-13 2014-10-08 株式会社ジーンデザイン Novel oligonucleotide derivative and NF-κB decoy comprising the same
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Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2675803B1 (en) * 1991-04-25 1996-09-06 Genset Sa CLOSED, ANTISENSE AND SENSE OLIGONUCLEOTIDES AND THEIR APPLICATIONS.
GB2273932A (en) 1992-11-24 1994-07-06 Stiefel Laboratories Stable oligonucleotides
FR2703053B1 (en) * 1993-03-26 1995-06-16 Genset Sa STAPLE AND SEMI-STAPLE OLIGONUCLEOTIDES, PREPARATION METHOD AND APPLICATIONS.
KR20000065690A (en) * 1999-04-08 2000-11-15 박종구 Specific and stable antisense oligonucleotide, antisense DNA and process for preparation thereof

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